Pathway of glutamate oxidation and its regulation in the HuH13 line of human hepatoma cells.

Well-coupled mitochondria were isolated from a HuH13 line of human hepatoma cells and human liver tissue. The liver mitochondria showed a feeble glutamine oxidation activity in contrast to the hepatoma mitochondria, whereas they utilized glutamate well for the oxidative phosphorylation. In the liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via transamination pathway in the tumor mitochondria. In the liver mitochondria, bicarbonate nearly at a physiological concentration inhibited oxygen uptake with glutamate as substrate. The interaction of bicarbonate with the pathway of glutamate oxidation occurred primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by the compound. In contrast to the liver mitochondria, glutamate oxidation was not affected by bicarbonate in the tumor mitochondria. These results indicate that the aberrations in the glutamate metabolism and its regulation observed in the hepatoma mitochondria may be favorable to the respiration utilizing glutamine and/or glutamate as an energy source.     

Properties of mitochondria from cells of the "fermentative" variant of Endomyces magnusii]

The properties of mitochondria from the cells of the "fermentative" variant of End. magnusii were studied. The induced fermentative transformation was brought about by a non-balanced vitamin cultivation. It was shown that the "fermentative" variant of End. magnusii represents an interesting model, in which the energy required for the cell functioning is provided for by a high fermentative activity and a normally functioning respiratory chain. The "fermentative" variant mitochondria were tightly coupled and possessed theoretical efficiency during oxidation of NAD-dependent substrates, which suggested the existence of all the three sites of energy coupling and phosphorylation at the substrate level. A specificity of energy regulation of the End. magnusii "fermentative" variant mitochondria, e. g. tight coupling during oxidation of succinate and lack of tight coupling during oxidation of exogenous NADH, is discussed. The tight coupling during succinate oxidation is confirmed by the observation of reverse electron transfer. Thus, the energy-dependent reduction of NAD during succinate oxidation has been firstly demonstrated for the mitochondria of yeast grown on a fermentable substrate.     

Ferrous-iron induces lipid peroxidation with little damage to energy transduction in mitochondria

Addition of ferrous sulfate, but not ferric chloride, in micromolar concentrations to rat liver mitochondria induced high rates of consumption of oxygen. The oxygen consumed was several times in excess of the reducing capacity of ferrous-iron (O: Fe ratios 5–8). This occurred in the absence of NADPH or any exogenous oxidizable substrate. The reaction terminated on oxidation of ferrous ions. Malondialdehyde (MDA), measured as thiobarbituric acid-reacting material, was produced indicating peroxidation of lipids. The ratio of O₂: MDA was about 4: 1. Pretreatment of mitochondria with ferrous sulfate decreased the rate of oxidation (state 3) with glutamate (+malate) as the substrate by about 40% but caused little damage to energy tranduction process as represented by ratios of ADP: O and respiratory control, as well as calcium-stimulated oxygen uptake and energy-dependent uptake of [⁴⁵Ca]-calcium. Addition of succinate or ubiquinone decreased ferrous iron-induced lipid peroxidation in intact mitochondria. In frozen-thawed mitochondria, addition of succinate enhanced lipid peroxidation whereas ubiquinone had little effect. These results suggest that ferrous-iron can cause peroxidation of mitochondrial lipids without affecting the energy transduction systems, and that succinate and ubiquinone can offer protection from damage due to such ferrous-iron released from the stores within the cells.     

Cyclic AMP activation of respiration of the liver mitochondria in different metabolic states]

cAMP 10(-6) activates the liver mitochondria respiration in all the metabolic states and failed to change or increased the phosphorylation rate in the oxidation of saturating concentration of succinate and isocitrate. Preincubation of mitochondria or homogenate of the liver with cAMP is obligatory for this effect. The fraction V of serum albumin and EDTA did not prevent the effect. Noradrenaline enhanced the mitochondrial respiration only in incubation with the homogenate. The effect of noradrenaline and cAMP was not summed up. Probably the noradrenaline effect was mediated through cAMP. The data obtained are against the decisive role of the respiration and phosphorylation uncoupling or the oxidation substrate accumulation and lead to the assumption on the mitochondria enzymes activation.     

Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles.

Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.     

Inhibition of oxidative phosphorylation by organotin thiocarbamates

A series of triphenyl-, tricyclohexyl- and tribenzyltin compounds have been synthesized and examined as inhibitors of mitochondrial oxidative phosphorylation. All compounds tested inhibit oxidative phosphorylation linked to succinate oxidation by potato tuber mitochondria. All of the organotin compounds inhibit ADP-stimulated O2 uptake linked to succinate oxidation with concentrations for 50% inhibition in the range 2-50 microM. This inhibition is not due to inhibition of electron transport from succinate to O2 per se: none of the organotin compounds at 50 microM substantially inhibit the rate of succinate oxidation in the presence of 2,4-dinitrophenol. Representative organotin compounds at 0.5-50 microM do not act as uncouplers of succinate oxidation. It is concluded that the organotin compounds act as energy transfer inhibitors to inhibit oxidative phosphorylation in potato tuber mitochondria. A similar mode of action of representative organotin compounds was found with rat liver mitochondria. These organotin compounds inhibit a hydrophobic Ca2+-dependent plant protein kinase in the absence but not in the presence of thiols.     

Disturbance of Oxidative Phosphorylation in the Mitochondria of Late Post-Traumatic Epileptic Nidus

We measured the rate of oxygen consumption by the mitochondria from the brain tissues of rabbits within a remote period after light cranio-cerebral trauma. One and six months after traumatization, oxidative phosphorylation in rabbits of the experimental groups demonstrated no significant difference from that in the control group. Yet, after a 12-month-long interval, clear differences were observed within the cortical zone with post-traumatic epileptic nidus. The coefficient of energy production decreased, and the process of oxidative phosphorylation became uncoupled. When succinate was used as a substrate for oxidation, we observed significant decreases in the rate of oxygen consumption in ADP phosphorylation and in the coefficient of respiration control. A significant decrease in the rate of oxygen consumption in the resting state (V ₂), the absence of disturbances in the respiration control, and preservation of a sufficient reserve ATPase activity were characteristic features when glutamate was used as a substrate. It seems probable that such shifts in oxidative phosphorylation can result in creation of an excessive glutamate pool and provide excessive epileptogenic glutamatergic activation of the neurons.     

STRUCTURAL CHANGES IN ISOLATED LIVER MITOCHONDRIA OF RATS DURING ESSENTIAL FATTY ACID DEFICIENCY

Liver mitochondria isolated in 0.44 M sucrose from rats deficient in essential fatty acids (EFA) oxidized citrate, succinate, α-ketoglutarate, glutamate, and pyruvate at a faster rate than did mitochondria isolated from normal rats; however, the oxidation of malate, caprylate, and β-hydroxybutyrate was not significantly increased. The mitochondria from deficient rats exhibited an increased ATPase activity and extensive structural damage as revealed by electron microscope examination of thin sections. An increase in citrate oxidation and ATPase activity, together with some structural damage, could be demonstrated as early as the 4^th week in rats on a fat-free diet. Saturated fat in the diet did not prevent the change in mitochondrial structure but accelerated its appearance. Both the biochemical and structural defects could be reversed within three weeks after feeding deficient rats a source of EFA. In the presence of a phosphate acceptor the effect of EFA deficiency on substrate oxidation was largely eliminated. A trend toward a reduced efficiency of oxidative phosphorylation was noted in mitochondria from EFA-deficient rats, but significant uncoupling was found only in the case of citrate, β-hydroxybutyrate, and glutamate in the presence of malonate. Together with the increased ATPase activity, the uncoupling of phosphorylation could account for the poor respiratory control found with the deficient preparation. However, EFA deficiency was without effect on the respiration of liver slices, which supports the belief that the observed changes in oxidation and phosphorylation are an artifact resulting from damage sustained by the deficient mitochondria during their isolation.     

Mode of Action of Antibiotic U-24,544

Antibiotic U-24,544, a new antibacterial agent, was found to be an effective uncoupler of phosphorylation associated with the oxidation of glutamate and succinate in rat liver mitochondria. Respiration was inhibited during glutamate oxidation but not during succinate oxidation. In a medium deficient in inorganic phosphate, the agent showed slight stimulation of mitochondrial glutamate oxidation. Mitochondrial swelling induced by inorganic phosphate was suppressed. The antibiotic inhibited protein, nucleic acid, and cell wall synthesis in Mycobacterium avium cells nearly equally without a predominant inhibition of any one of these macromolecular biosynthetic processes. Nucleic acid and polypeptide synthesis remained unaffected, but respiration was inhibited in cell-free bacterial systems. It was thus concluded that the antibiotic interfered primarily with the cellular energy-generating processes.     

Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.     

Action of different nucleotides on the last step of aldosterone synthesis

The effect of various nucleotides on the last step of aldosterone biosynthesis, the so-called "18 oxidation" (transformation of 18-hydroxycorticosterone to aldosterone), was studied by incubation of tritiated 18-hydroxycorticosterone with untreated duck adrenal mitochondria in vitro. The study was carried out in the absence or in the presence of antimycin A which blocks the respiratory chain. Results show that, when oxidative phosphorylation chain functions normally, GTP and CTP had no effect, UTP stimulated this reaction but ADP and ATP inhibited the transformation of 18-hydroxycorticosterone into aldosterone to the same extent. For this reason ATP is included in all controls for experiments studying the effect of ATP when "18 oxidation" is inhibited by antimycin A. When oxidative phosphorylation chain is inhibited by antimycin A, ATP is able to reverse the inhibition of "18 oxidation" induced by antimycin A, in the presence of succinate. Under these conditions UTP is not able to reverse the inhibition induced by antimycin A; GTP and CTP had no effect. Effects of ATP and UTP on the last step of aldosterone biosynthesis are related to different mechanisms. ATP clearly acts as an energy source for "18 oxidation" in the presence of succinate. The role of UTP must still be determined.     

Effects of phthalate esters on the respiration of rat liver mitochondria.

The effects of phthalate esters on the oxidation of succinate, glutamate, beta-hydroxybutyrate and NADH by rat liver mitochondria were examined and it was found that di-n-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NAD-linked substrates such as glutamate and beta-hydroxybutyrate. However, oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.     

The respiration and oxidative phosphorylation of the brain mitochondria in rats with different types of behavior]

Respiration and oxidative phosphorylation of brain mitochondria were studied in outbred rats with different types of behaviour in open-field test and in forced swimming test. It was found that in rats with "active" type of behaviour (with high locomotor activity and low level of depressiveness) the brain mitochondria have at succinate oxidation a higher rate of respiration in active metabolic state and in presence of uncoupler 2,4-DNP, and a higher rate of ADP phosphorylation than in rats with "passive" type of behaviour (with low locomotor activity and high and moderate levels of depressiveness). Thus rats with active type of behaviour have a higher succinate oxidase activity than rats with passive type of behaviour. It is supposed that revealed differences in brain energy metabolism may affect the dominance of certain type of behaviour.     

Oxaloacetate inhibition of succinate oxidation in tightly coupled liver mitochondria with ferricyanide as an electron acceptor

When ferricyanide is used as an artificial electron acceptor, succinate oxidation by tightly coupled liver mitochondria becomes inhibited after 1–3 min. No inhibition occurs in the presence of rotenone or glutamate establishing that oxaloacetate causes the inhibtion. Oxygen consumption by mitochondria oxidizing succinate does not become inhibited in the absence of rotenone suggesting that oxaloacetate accumulates to a greater extent when ferricyanide is added than when oxygen is the terminal acceptor. Higher levels of oxaloacetate in the ferricyanide reaction are apparently due to an increased rate of synthesis rather than a decreased rate of removal. Thus it appears that when succinate is the substrate and oxygen the terminal acceptor a control mechanism exists which blocks oxidation of malate. When ferricyanide is added as an artificial electron acceptor this control is lost and oxaloacetate accumulates to inhibit succinate oxidation.     

Amaranth seed oil: Effect of oral administration on energetic functions of rat liver mitochondria activated with adrenaline

Respiration parameters of liver mitochondria (MCh) in rats fed with amaranth seed oil for 3 weeks have been evaluated. Thirty minutes before decapitation, adrenaline was injected intraperitoneally at a low dose (350 μg/kg body weight) to both control and experimental animals. It was shown that in animals that were injected with adrenaline and did not receive oil, the rate of phosphorylating respiration increased by 32% and phosphorylation time decreased by 22% upon oxidation of succinate; upon oxidation of α-ketoglutarate in the presence of the succinate dehydrogenase inhibitor malonate, phosphorylating respiration was activated by 23%. The respiration of MCh upon oxidation of succinate + glutamate and α-ketoglutarate in the absence of malonate was not affected by adrenaline. The intake of oil markedly activated almost all parameters of mitochondrial respiration in experimental rats upon oxidation of all above-listed substrates in both coupled and uncoupled MCh. However, phosphorylation time was close to the control value (upon oxidation of succinate) or increased (upon oxidation of α-ketoglutarate in the presence and absence of malonate). The injection of adrenaline to animals receiving oil did not affect the oil-activated respiration of MCh oxidizing the substrates used; however, phosphorylation time in all groups of animals decreased. Ca²⁺ capacity of MCh in rats receiving amaranth oil did not change. Thus, our data show that feeding of rats with amaranth oil activates mitochondrial respiration and prevents MCh hyperactivation induced by adrenaline.     

Changes in bioenergetics of skeletal muscle mitochondria during experimental trichinosis in rats

Changes in bioenergetics were studied in mitochondria isolated from skeletal muscles of rats in the course of experimental trichinosis. In fortified media supporting oxidative phosphorylation, oxygen uptake with succinate was greatly enhanced, while the oxidation of glutamate and of malate plus pyruvate was manifold inhibited. The protective effect of BSA added was negligible in this case.     

Hydrogen peroxide generation by higher plant mitochondria oxidizing complex I or complex II substrates.

The generation of H2O2 by isolated pea stem mitochondria, oxidizing either malate plus glutamate or succinate, was examined. The level of H2O2 was almost one order of magnitude higher when mitochondria were energized by succinate. The succinate-dependent H2O2 formation was abolished by malonate, but unaffected by rotenone. The lack of effect of the latter suggests that pea mitochondria were working with a proton motive force below the threshold value required for reverse electron transfer. The activation by pyruvate of the alternative oxidase was reflected in an inhibition of H2O2 formation. This effect was stronger when pea mitochondria oxidized malate plus glutamate. Succinate-dependent H2O2 formation was ca. four times lower in Arum sp. mitochondria (known to have a high alternative oxidase) than in pea mitochondria. An uncoupler (FCCP) completely prevented succinate-dependent H2O2 generation, while it only partially (40-50%) inhibited that linked to malate plus glutamate. ADP plus inorganic phosphate (transition from state 4 to state 3) also inhibited the succinate-dependent H2O2 formation. Conversely, that dependent on malate plus glutamate oxidation was unaffected by low and stimulated by high concentrations of ADP. These results show that the main bulk of H2O2 is formed during substrate oxidation at the level of complex II and that this generation may be prevented by either dissipation of the electrochemical proton gradient (uncoupling and transition state 4-state 3), or preventing its formation (alternative oxidase). Conversely, H2O2 production, dependent on oxidation of complex I substrate, is mainly lowered by the activation of the alternative oxidase.     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Effects of chronic ethanol treatment of mitochondrial functions damage to coupling site I

Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg²⁺- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD⁺-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD⁺-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the $\text{P}\text{O}$ ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD⁺-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca²⁺ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD⁺-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.     

Different effects of 2,4-dinitrophenol on rat liver mitochondrial oxidation of various substrates: succinate and glutamate vs 3-hydroxybutyrate and glycerol 3-phosphate

In homogenate and isolated mitochondria from the liver of male and female rats of Wistar and August strains, a higher uncoupled vs State 3 oxidation of succinate and glutamate and lower that of 3-hydroxybutyrate and glycerol 3-phosphate were observed. The optimal concentration of 2,4-dinitrophenol (DNP) was in the range 7-12 microM for 3-hydroxybutyrate oxidation and 50-100 microM for succinate or glutamate oxidation.